Charged compounds comprising a nucleic acid binding moiety and uses therefor

ABSTRACT

Charged compounds are provided that comprise one or more regions of localized positive charge, compositions comprising such compounds, methods of synthesizing such compounds, methods of screening such compounds to identify those having anti-infective activity, and methods of using such compounds to prevent or inhibit infections. These compounds, and compositions containing them, have multiple applications, including use in human and animal medicine and in agriculture.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Ser. No. 60/189,930,filed Mar. 16, 2000, the disclosure of which is incorporated herein byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] The United States government may have certain rights to thisinvention pursuant to DARPA grant no. N65236-99-1-5427.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK BACKGROUND OF THE INVENTION

[0003] Many compounds, either naturally occurring or synthetic, havebeen found to bind to double stranded nucleic acid, especially doublestranded deoxyribonucleic acid (“dsDNA”). Depending on their structure,the compounds bind to different parts of the nucleic acid. Some bind tothe major groove while others associate with the minor groove. Stillothers intercalate between adjacent base pairs. Combination bindingmodes are also known, in which a compound has binding interactions withmore than one site in the nucleic acid.

[0004] Certain dsDNA binding compounds may be used to regulate theexpression of genes for medical purposes. If a disease is characterizedby the overexpression or the undesired expression of a gene (e.g., anoncogene), the disease may be treated by suppressing in toto or in partthe expression of the gene by the binding of such compounds to the geneor a promoter site thereof. Infections by pathogens such fungi,bacteria, and viruses may be combated with compounds that affect theexpression of genes essential for the proliferation of the pathogen.

[0005] Whatever the application, the compound must strongly bind todsDNA, generally meaning that it binds with an association constant ofat least 10⁶ M⁻¹, preferably at least about 10⁹ M⁻¹. However, bindingstrength alone is not determinative of efficacy. Many other factors comeinto play, including, for instance, cellular uptake, stability,toxicity, binding specificity, and the like. A compound that isacceptable or superior in one characteristic may be fatally deficient inanother characteristic. Thus, there is a continuing need to develop newclasses of nucleic acid binding compounds for use in such applications.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a new class of compounds, as wellas compositions comprising such compounds, methods of synthesizing suchcompounds, methods of screening such compounds to identify those havinganti-infective activity, and methods of using such compounds to preventor inhibit infections.

[0007] In one aspect, the invention provides a class of chargedcompounds. The members of this class of compounds each comprise anucleic acid binding moiety, and can be represented by formula (I):

W—Y-[Het]-L-[NABM]  (I)

[0008] or a salt thereof, preferably a pharmaceutically acceptable salt.Additionally, esters, amides, prodrugs, isomers, or metabolites offormula I are also within the scope of the present invention.

[0009] With respect to this invention, “NABM” refers to nucleic acidbinding moiety, particularly nucleic acid binding moieties that bind toor associate with double-stranded nucleic acids, particularly dsDNA.NABMs include small molecules, proteins, and nucleic acids. Preferredsmall molecules include polyamides, particularly synthetic polyamides,and preferred nucleic acids include oligonucleotides. NABMs includeintercalating moieties, minor groove binding moieties, major groovebinding moieties, and those that include moieties that bind in acombination of such modes, e.g., an NABM that includes both minor andmajor groove binding moieties.

[0010] In formula I above, an NABM is linked to a heteroaromatic moiety(“Het”) via linker “L”. L represents a bond, preferably a covalent bond,or a linking group. Het represents a heteroaromatic moiety other thanN-methyl or N-hydrogen pyrrole, selected from the group consisting of

[0011] wherein one of X₁, X₂, and X₃ is a ring vertex selected from thegroup consisting of —O—, —S—, and —NR₃—, and the other two of X₁, X₂,and X₃ are ring vertices selected from the group consisting of ═N— and═CR₄—.

[0012] Covalently attached to the heteroaromatic moiety [Het] is asubstituent having the formula:

W—Y-,

[0013] wherein Y is selected from O, S, S(O), SO₂, C(R₁)₂, N(R₃)SO₂,SO₂N(R₃) and NR₃; and W is halogen or a group having the formula:

[0014] The various R groups in formula I have the following meanings:each R₁ is independently selected from H, F, substituted orunsubstituted (C₁-C₆)alkyl and a substituted or unsubstituted(C₁-C₆)heteroalkyl group; R₂ is a moiety bearing a polar group if Y isother than NR₃ and is a moiety bearing a polar group, a substituted orunsubstituted (C₁-C₁₂)alkyl group or a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group if Y is NR₃; each R₃ is independently selectedfrom H, a substituted or unsubstituted (C₁-C₁₂)alkyl group and asubstituted or unsubstituted (C₁-C₁₂)heteroalkyl group, provided thatneither of R₂(R₁)₂C and R₃ contains a 2-chloroethyl or 2-hydroxyethylgroup when Y equals NR₃; and each R₄ is independently selected fromhydrogen, halogen, an amino group, a (C₁-C₈)alkylamino group, adi(C₁-C₈)alkylamino group, a tri(C₁-C₈)alkyl ammonium group, a hydroxylgroup, a (C₁-C₈)alkoxy group, a thiol group, a (C₁-C₈)thioether group, a(C₁-C₈)sulfone group, a (C₁-C₈)sulfoxide group, a (C₁-C₈)sulfonamidegroup, a substituted or unsubstituted (C₁-C₁₂)alkyl group and asubstituted or unsubstituted (C₁-C₁₂)heteroalkyl group.

[0015] Additionally, at least one of R₂, [Het], or [NABM] has a positivecharge.

[0016] In one group of preferred embodiments, the compounds of theinvention can be represented by formula (Ia)

[0017] or a salt, preferably a pharmaceutically acceptable salt, or anester, amide, prodrug, isomer, or metabolite thereof.

[0018] In formula Ia, the subscripts a, b, and d are each independently0, 1, 2, 3, 4, or 5, with the proviso that at least one of a, b, or d isother than 0. The subscripts c, e and f are each independently 0 or 1.

[0019] In these embodiments, R₅ is selected from halogen, OR₇ andN(R₇)₂. R₆ is selected from hydrogen, halogen, a substituted orunsubstituted (C₁-C₁₂)alkyl group and a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group. Each R₇ is independently selected fromhydrogen, a substituted or unsubstituted (C₁-C₁₂)alkyl group and asubstituted or unsubstituted (C₁-C₁₂)heteroalkyl group. Each Q isindependently selected from —(CH₂)₂—, —(CH₂)₃—, and a heteroaromaticring independently selected from the group consisting of substituted orunsubstituted imidazole, pyrrole, pyrazole, furan, isothiazole, oxazole,isoxazole, thiazole, furazan, 1,2,3-thiadiazole, 1,2,4-thiadiazole,1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole;1,2,4-oxadiazole, 1,3,4-oxadiazole, and thiophene rings. Preferably, Qis a thiophene ring. Exemplary suitable substituents in a heteroaromaticring Q include Cl, F, CH₃, and hydroxy.

[0020] Compounds according to the invention can be in unpurified,substantially purified, and purified forms. The compounds can be presentwith any additional component(s) such as a solvent, reactant, orby-product that is present during compound synthesis or purification,and any additional component(s) that is present during the use ormanufacture of a compound or that is added during formulation orcompounding of a compound.

[0021] In another aspect, the present invention provides methods forsynthesizing the compounds of the invention. Broadly, such methodscomprise linking a NABM to R₂(R₁)₂C—Y-[Het], either directly or throughan optional linking group L. The various moieties of the invention canbe synthetic or natural products. Synthetic moieties may be synthesizedby solution or solid phase methods. Two or moieties may also besynthesized together.

[0022] In yet another aspect, the invention provides compositionscomprising a compound according to the invention and one or moreexcipients, diluents, or carriers. Such compositions can be dry orliquid formulations. The particular composition employed will depend onthe intended application for the compound. Compounds according to thisinvention have been found to be strongly bind dsDNA. Preferably, theassociation constant for a compound of the invention an dsDNA is atleast about 10⁶ M⁻¹, more preferably at least about 10⁹ M⁻¹, and mostpreferably about 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹ or more. Some compositionshave been found to be effective in inhibiting the proliferation ofpathogens such as fungi and bacteria.

[0023] Applications for the compounds and compositions of the inventioninclude anti-infective uses. Such uses can be prophylactic ortherapeutic in nature. These uses are accomplished by contacting apathogen of a eukaryotic organism with an amount of a compound of theinvention sufficient to achieve the desired result. Contacting can occurin vitro or in vivo, as the context requires. Preferred embodiments ofthis aspect involve inhibiting the proliferation of a pathogenicorganism. Inhibition can be achieved by killing the organism, byreducing its rate of proliferation, or by reducing or eliminating apathogenic aspect of the organism, for example, by inhibiting expressionof a pathogenic gene (e.g., a gene encoding a toxin). Representativepathogens that can be affected by the preventative and therapeuticmethods of the invention include eukaryotic and prokaryotic organisms,as well as viruses. Preferred targets are bacteria and fungi.

[0024] The treatment-related aspect of this invention is directed toboth animals and plants that serve as hosts, or intermediaries, for thetargeted pathogen. As such, the invention has implications in animalhealth and medicine as well as in agriculture.

[0025] In yet another aspect, the invention provides methods ofscreening to identify compounds of the invention that haveanti-infective activity. These screening methods include both in vitroand in vivo screening methods, and can include methods involving an invitro screen followed by an in vivo screen (e.g., a cell-based screen).In either format, the methods are preferably high throughput methods,meaning that more than about 10, preferably, more than about 100, 1,000,or 10,000 compounds are screened at once. In each of the aboverecitations, a “charged compound” refers to compounds that arepositively charged under assay or physiological conditions, which aretypically neutral or slightly acidic (pH about 5 to about 7). Manycompounds are illustrated as having amine components in their neutralform. Nevertheless, one of skill in the art will appreciate that theseamines can carry a positive charge (e.g., be protonated) atphysiological pH or under typical assay conditions.

[0026] These and other aspects and embodiments of the invention aredescribed further in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Not applicable

DETAILED DESCRIPTION OF THE INVENTION

[0028] Abbreviations and Definitions

[0029] The term “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers.

[0030] The term “alkylene” by itself or as part of another substituentmeans a divalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingsix or fewer carbon atoms.

[0031] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy)are used in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

[0032] The term “heteroalkyl,” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and from one to threeheteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S may be placed at any interior position of the heteroalkyl group.The heteroatom Si may be placed at any position of the heteroalkylgroup, including the position at which the alkyl group is attached tothe remainder of the molecule. Examples include —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂— and -CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

[0033] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves orin combination with other terms, represent, unless otherwise stated,cyclic versions of “alkyl” and “heteroalkyl”, respectively.Additionally, for heterocycloalkyl, a heteroatom can occupy the positionat which the heterocycle is attached to the remainder of the molecule.Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

[0034] The terms “halo” or “halogen,” by themselves or as part ofanother substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom. Additionally, terms such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. Forexample, the term “halo(C₁-C₄)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0035] The term “aryl” means, unless otherwise stated, apolyunsaturated, typically aromatic, hydrocarbon substituent which canbe a single ring or multiple rings (up to three rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from zero to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

[0036] For brevity, the term “aryl” when used in combination with otherterms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl andheteroaryl rings as defined above. Thus, the term “arylalkyl” is meantto include those radicals in which an aryl group is attached to an alkylgroup (e.g., benzyl, phenethyl, pyridylmethyl and the like) includingthose alkyl groups in which a carbon atom (e.g., a methylene group) hasbeen replaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0037] Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

[0038] Substituents for the alkyl and heteroalkyl radicals (includingthose groups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —N′—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2m′+1),where m′ is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Preferably, thesubstituted alkyl and heteroalkyl groups have from 1 to 4 substituents,more preferably 1, 2 or 3 substituents. Exceptions are those perhaloalkyl groups (e.g., pentafluoroethyl and the like) which are alsopreferred and contemplated by the present invention.

[0039] Similarly, substituents for the aryl and heteroaryl groups arevaried and are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′,—R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′, —NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

[0040] Two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —T—C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—,—O—, —CH₂— or a single bond, and q is an integer of from 0 to 2.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—,—NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 3. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

[0041] As used herein, the term “heteroatom” is meant to include oxygen(O), nitrogen (N), sulfur (S) and silicon (Si).

[0042] The term “pharmaceutically acceptable salts” is meant to includesalts of the active compounds which are prepared with relativelynontoxic acids or bases, depending on the particular substituents foundon the compounds described herein. When compounds of the presentinvention contain relatively acidic functionalities, base addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

[0043] The neutral forms of the compounds may be regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

[0044] In addition to salt forms, the present invention providescompounds which are in a prodrug form. Prodrugs of the compoundsdescribed herein are those compounds that readily undergo chemicalchanges under physiological conditions to provide the compounds of thepresent invention. Additionally, prodrugs can be converted to thecompounds of the present invention by chemical or biochemical methods inan ex vivo environment. For example, prodrugs can be slowly converted tothe compounds of the present invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent.

[0045] Certain compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areintended to be encompassed within the scope of the present invention.Certain compounds of the present invention may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated by the present invention and areintended to be within the scope of the present invention.

[0046] Certain compounds of the present invention possess asymmetriccarbon atoms (optical centers) or double bonds; the racemates,diastereomers, geometric isomers and individual isomers are all intendedto be encompassed within the scope of the present invention.

[0047] The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

[0048] In the discussions below, reference is made to dsDNA as thenucleic acid, but it is to be understood that the invention is notlimited to dsDNA and is applicable to other nucleic acids, i.e.,ribonucleic acid.

[0049] Compounds.

[0050] Nucleic acid binding compounds of this invention represented byformula (I) (reproduced below for convenience)

W—Y-[Het]-L-[NABM]  (I)

[0051] are now discussed in greater detail, especially with reference topreferred embodiments thereof.

[0052] As noted above, [NABM] is a double-stranded nucleic acid bindingmoiety; L is a covalent bond or a linking group; [Het] is aheteroaromatic moiety other than N-methyl or N-hydrogen pyrrole,selected from:

[0053] wherein one of X₁, X₂, and X₃ is a ring vertex selected from —O—,—S—, and —NR₃—, and the other two of X₁, X₂, and X₃ are ring verticesselected from ═N— and ═CR₄—. Additionally, the circle in thefive-membered ring above is meant to indicate the presence of two doublebonds, which, in some embodiments, can move within the ring.

[0054] Returning to formula I, the letter Y represents O, S, S(O), SO₂,C(R₂)₂, N(R₃)SO₂, SO₂N(R₃), or NR₃. The letter W represents a halogenatom of a group having the formula:

[0055] The various R groups have the following meanings: each R₁ isindependently selected from H, F, substituted or unsubstituted(C₁-C₆)alkyl and a substituted or unsubstituted (C₁-C₆)heteroalkylgroup; R₂ is a moiety bearing a polar group if Y is other than NR₃ andis a moiety bearing a polar group, a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group or a substituted or unsubstituted(C₁-C₁₂)alkyl group if Y is NR₃; each R₃ is independently selected fromH, a substituted or unsubstituted (C₁-C₁₂)alkyl group, and a substitutedor unsubstituted (C₁-C₁₂)heteroalkyl group, provided that neither ofR₂(R₁)₂C and R₃ contains a 2-chloroethyl or 2-hydroxyethyl group when Yequals NR₃; and each R₄ is independently selected from hydrogen,halogen, an amino group, a (C₁-C₈)alkylamino group, adi(C₁-C₈)alkylamino group, a tri(C₁-C₈)alkyl ammonium group, a hydroxylgroup, a (C₁-C₈)alkoxy group, a thiol group, a (C₁-C₈)thioether group, a(C₁-C₈)sulfone group, a (C₁-C₈)sulfoxide group, a (C₁-C₈)sulfonamidegroup, a substituted or unsubstituted (C₁-C₁₂)alkyl group and asubstituted or unsubstituted (C₁-C₁₂)heteroalkyl group.

[0056] Additionally, at least one of R₂, [Het], or [NABM] has a positivecharge. In preferred embodiments, at least two of of R₂, [Het], or[NABM] has a positive charge. In still other preferred embodiments, atleast one of R₂, [Het], or [NABM] carries two or more positive charges.Accordingly, in a particularly preferred embodiment, at least two amongR₂, the heteroaromatic moiety [Het], and the NABM have a positivecharge, so that compound (I) overall has at least two positive charges.Where it is stated that R₂, Het, or NABM has a positive charge, it doesnot mean that R₂ (or Het or NABM, as the case may be) is limited tohaving a single positive charge; multiple positive charges are alsocontemplated. Further, the state of being positively charged is in thecontext of approximately neutral aqueous or substantially aqueousconditions (e.g., a small amount of an organic solvent may be present),preferably under physiological conditions, i.e., a set of parametersthat describe an intracellular (e.g., periplasm or cytoplasm) orextracellular environment. Such parameters include pH, temperature,ionic composition and strength, buffering capacity, etc., and these willvary depending upon various factors, including the host organism (e.g.,animal or plant), the environment in which the compound is to bedelivered, for example, into the blood of an animal or onto or into thesoil in which a crop plant is, or is intended to, grow. A positivecharge may result from the protonation of an amine, amidine, orguanidine group, or a less basic group such as a pyridine, pyridazine,pyrimidine, pyrazine, imidazole, or aniline group. Depending on suchgroup's ionization constant, it may be substantially fully protonated oronly partially protonated. Generally, as a matter of convenience, agroup or moiety that may become positively charged by protonation isdepicted in the structural formulae herein in its unprotonated form.

[0057] In other preferred embodiments, Y is NR₃ and R₃ is either H orlower alkyl. R₁ preferably is hydrogen. Exemplary suitable (C₁-C₁₂)alkylor aryl groups for R₂, R₃, or R₄ include methyl, ethyl, propyl,isopropyl, n-butyl, s-butyl, isobutyl, cyclopentyl, cyclohexyl, phenyl,C₆H₁₁CH₂, C₆H₅CH₂, C₅H₉CH₂, and the like. In one group of preferredembodiments, the C₁-C₁₂ alkyl group is substituted with at least onesubstituent selected from (C₁-C₄)alkene group, a (C₁-C₄)alkyne group, anamino group, a (C₁-C₈)alkylamino group, a di(C₁-C₈)alkylamino group, atri(C₁-C₈)alkyl ammonium group, a hydroxy group, a (C₁-C₈)alkoxy group,a thiol group, a (C₁-C₈)thioether group, a (C₁-C₈)sulfone group, a(C₁-C₈)sulfoxide group, a (C₁-C₈)sulfonamide group, a (C₁-C₈)acyl group,a mono or di(C₁-C₈) N-alkylamide group, a thiol group, a(C₁-C₄)thioether group, a (C₁-C₄)sulfone group, a (C₁-C₄)sulfoxidegroup, a mono or di(C₁-C₈) N-alkylsulfonamide group, a halogen, a(C₃-C₇)cycloaliphatic group, a five-, six- or seven-memberedheterocyclic group, an aryl group, and a heteroaryl group. In someembodiments, the (C₁-C₁₂)alkyl group is substituted with two, three orfour of the functional group components provided above.

[0058] Returning to formula I, the polar group of R₂ can be a positivelycharged group such as a protonated primary, secondary, or tertiary aminogroup or a quaternary ammonium group. In some embodiments, the polargroup will not be positively charged. Preferred examples of unchargedpolar groups include hydroxy, cyano, fluoro, ether, ketone, sulfonamido,sulfone, and carboxamido groups, although other suitable polar groupsmay be employed. Here, a polar region is one that has a dipole momentgreater than that of a C—C or C—H covalent bond.

[0059] For those preferred embodiments in which Y is NR₃, the R₂ groupneed not contain a polar group. In such instances, R₂ may be equal toR₃—that is the partial formula R₂(CR₁)₂Y reduces to R₃(CR₁)₂NR₃, wherethe two R₃'s are independently variable. The two R₃'s may be joined toform a ring structure, preferably containing 4, 5, 6, or 7 atoms. Thering may contain heteroatom moieties, such as —NH—, —NMe-, —O—, —N(loweralkyl)-, —S—, —SO₂—, —SO—, and the like as part of the ring. The ringalso may contain substituents pendant therefrom. Suitable substituentsare provided in the definitions of alkyl and aryl substituents, above.

[0060] For those embodiments in which NABM is positively charged, thepositive charge may be situated in a moiety pendant from the side ofNABM (i.e., from an internal heteroaromatic or aliphatic moiety) and/orin a terminal moiety distal from Het. The positively charged moiety maybe derived from a basic amino acid (e.g., lysine, histidine, orarginine) or a peptide unit comprising one or more basic amino acids. Apreferred peptide configuration is one in which a proline separates twobasic amino acids (e.g., Arg-Pro-Arg). Exemplary suitable positivelycharged moieties are disclosed in Dervan et al., WO 98/37087 (1998) andRothbard et al., WO 98/52614 (1998), the disclosures of which areincorporated herein by reference. As those in the art will appreciate,however, the NABM need not always have a positive charge, as illustratedby compound X (infra).

[0061] As described above, the nucleic acid binding moiety [NABM] may bea dsDNA intercalator, a dsDNA minor groove binding moiety, or a dsDNAmajor groove binding moiety. It is to be understood that, where [NABM]is referred to as a “minor groove binder” (or words to that effect), itdoes not mean that such moiety has binding interactions exclusively withthe minor groove; the moiety also may have binding interactions withother parts of the dsDNA, for example, with adjacent base pairs byintercalation, with backbone phosphate groups, or with the major groove.

[0062] [NABM] preferably is a minor groove binder, which typically (butnot necessarily) has an elongate crescent shape, topologicallycomplementary to the shape of the minor groove. The minor groove bindermay be a residue of a naturally occurring compound, such as doxorubicin,daunomycin, anthramycin, calicheamycin, mitomycin, CC-1065, duocarmycin,distamycin, and netropsin, or an analog or a derivative thereof.Alternatively, [NABM] may be a residue of a synthetic minor groovebinder, such as pentamidine, berenil, stilbamidine, DDUG, NSC 101327, SN6999, SN 6136, SN 16814, SN18071, NSC 57153, Hoechst 33258, Ionen X, andmethyl green, or an analog or a derivative thereof.

[0063] In particularly preferred embodiments, the nucleic acid bindingmoiety is a synthetic polyamide unit comprising N-methylpyrrolecarboxamide (“Py”) units and optionally one or more of N-methylimidazolecarboxamide (“Im”), N-methyl-3-hydroxypyrrole carboxamide (“Hp”),glycine carboxamide, β-alanine carboxamide, γ-aminobutyric acidcarboxamide, 5-aminovaleric acid carboxamide, and γ-2,4-diaminobutyricacid carboxamide units, such units being represented respectively by thestructures:

[0064] Some polyamides similar to those described herein have been shownto be minor groove binders, often binding with high binding constants(e.g., greater than 10⁹ M⁻¹). Disclosures relating to the design andsynthesis of such polyamides include Baird and Dervan, J. Am. Chem. Soc.118, 6141-6146 (1996) and U.S. application Ser. Nos. 08/607,078 (filedFeb. 26, 1996); 09/374,702 (filed Aug. 12, 1999); 09/372,473 (filed Aug.11, 1999); 09/372,474 (filed Aug. 11, 1999); 09/414,611 (filed Oct. 8,1999); and 09/479,279 (filed Jan. 5, 2000 and entitled “Compositions andMethods Relating to Cyclic Compounds that Undergo Nucleotide Base PairSpecific Interactions with Double Stranded Nucleic Acids”), thedisclosures of which are incorporated herein by reference. It has beenfurther discovered that such polyamides can bind to dsDNA with twoheteroaromatic carboxamide moieties fitting side-by-side within theminor groove and that such side-by-side heteroaromatic carboxamide pairsrecognize specific dsDNA base pairs, giving rise to a set of “pairingrules” correlating heteroaromatic carboxamide pairs and the DNA basepairs recognized: Heteroaromatic Pair dsDNA Base Pair(s) RecognizedIm/Py G/C Py/Im C/G Py/Py A/T, T/A Hp/Py T/A Py/Hp A/T

[0065] Accordingly, the pairing rules above can be used to design anNABM moiety that binds to dsDNA with specificity for particular basepair sequences.

[0066] Glycyl or β-alanyl carboxamides can serve as “spacer” groups foradjusting the position of the heteroaromatic carboxamide residues inrelation to the nucleotide base pairs of the NABM's binding site. Aγ-aminobutyric acid carboxamide, 5-aminovaleric acid carboxamide, orγ-2,4-diaminobutyric acid carboxamide unit (or other moieties thatproduce a substantially equivalent structural effect) introduces a“hairpin” into the polyamide and permits heteroaromatic carboxamideunits of the same NABM to bind side-by-side to each other. Use of twosuch units, for example, at each end of the NABM or at one end and at aninternal position, allows the formation of NABMs having otherconformations (e.g., cyclic or “H-pin” conformations, respectively). The2-amino group of γ-2,4-diaminobutyric acid provides an attachment pointfor tandem-linked polyamide units, as well as providing a moiety thatcan be used to introduce chirality into the NABM. A Py, Hp, or Pyequivalent heteroaromatic carboxamide may be replaced with a βcarboxamide to form pairs such as β/β or β/Py. These and other moleculardesign principles disclosed in the aforementioned references may be usedin the design of preferred examples NABM moieties of this invention.

[0067] In yet other embodiments, nucleic acid binding moiety [NABM]comprises the structure (II)

-Q₁-Z₁-Q₂-Z₂- . . . -Q_(m)-Z_(m)-

  (II)

[0068] where each of Q₁, Q₂, . . . , Q_(m) is a heteroaromatic moiety or(CH₂)_(p) and the subscript p is an integer from 1 to 3, inclusive; eachof Z₁, Z₂, . . . , Z_(m) is a covalent bond or a linking group; and thesubscript m is an integer from 1 to 9, inclusive, more preferably from 2to 4. For those embodiments in which Q is a heteroaromatic moiety, it ispreferably selected from optionally substituted imidazole, pyrrole,pyrazole, furan, isothiazole, oxazole, isoxazole, thiazole, thiophene,furazan, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole,1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, 1,3,4-oxadiazole,1,2,4-oxadiazole, and thiophene moieties. Exemplary substituents includeCl, F, CH₃ (e.g., as in N-methylpyrrole or N-methylimidazole), andhydroxy (e.g., as in 3-hydroxypyrrole).

[0069] Linking groups Z₁, Z₂, . . . Z_(m), are generally those divalentgroups having from 2 to 5 backbone atoms. The term “backbone” as appliedto linking groups in the present invention, refers to the atoms that arein a contiguous linkage between the two groups the atoms are joining.For example, two heteroaromatic moieties that are connected by —C(O)NH—are said to be linked by a group having two “backbone” atoms (e.g., thecarbon atom and the nitrogen atom). Exemplary linking groups includecarboxamide, amidine, and ester groups, as respectively illustratedbelow, with carboxamide groups being preferred:

[0070] In still other embodiments of the invention, the nucleic acidbinding moiety comprises the structure (III)

[0071] where each n is independently an integer from 1 to 9, inclusive(preferably n is 2 or 3), and each m is independently 0 or 1 (preferably1).

[0072] As noted above, the heteroaromatic moiety [Het] and NABM arejoined by a linking group L, which can be a covalent bond or a divalentlinking group having from 2 to 5 (preferably 2) backbone atoms.Exemplary linking groups include carboxamide, amidine, and ester groups,with carboxamide linking groups being preferred. Exemplary substituentsfor the [Het] groups include Cl, F, CH₃ (e.g., as in N-methylpyrrole orN-methylimidazole), and hydroxy (e.g., as in 3-hydroxypyrrole).

[0073] A variety of synthetic methods may be used to link Het and NABM.In addition to those described in the examples further below, othermethods known in the art may be used. Several such methods are citedhere for illustrative purposes, with a distamycin residue as the NABM.An amine-terminated distamycin residue may be alkylated with an epoxidecompound, as taught in Arcamone et al., U.S. Pat. No. 4,738,980 (1988)and U.S. Pat. No. 4,766,142 (1988). Another approach is provided inLazzari et al., U.S. Pat. No. 5,017,599 (1991); U.S. Pat. No. 5,049,579(1991); and U.S. Pat. No. 5,310,752 (1994) and Animati et al., U.S. Pat.No. 5,670,534 (1997): condensation of an acyl compound with anamino-terminated distamycin residue in the presence of a condensingagent such as dicyclohexylcarbodiimide to form a carboxamide linkage.Animati et al., U.S. Pat. No. 5,412,976 (1995) discloses the reaction ofa carboxyimidate with an amine-terminated distamycin residue to fom anamidine. The aforementioned patents are incorporated herein byreference.

[0074] The heteroaromatic moiety may be a substituted or unsubstitutedform of any of the following: imidazole, pyrrole other than N-methyl orN-hydrogen pyrrole, pyrazole, furan, isothiazole, oxazole, isoxazole,thiazole, furazan, 1,2,3-thiadiazole, 1,2,4-thiadiazole,1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole,1,2,4-oxadiazole, 1,3,4-oxadiazole, or thiophene moiety.

[0075] Preferably, Het is an isothiazole, as in

[0076] Preferred isothiazole-containing compounds according to thisinvention are represented by formula (IVa):

[0077] In compound (IVa), R₁, R₂, and R₄ are as previously defined, withreference to formula I.

[0078] Particularly preferred isothiazole-containing compounds of thisinvention comprise the structure (IVb)

[0079] where m, n, R₁, R₂, and R₄ are as previously defined.

[0080] In R₂(R₁)₂C—Y—, (shown more specifically in IVb as R₂(R₁)₂C—NH)the polar group(s) in R₂ (where present) are preferably one or moreprimary, secondary, or tertiary amino groups, which when protonated,make R₂ positively charged. Alternatively, the polar group is aquaternary ammonium group. In other preferred groups, each R₁ ishydrogen.

[0081] More preferably, R₂ has an amino group separated from the Y groupby about 2 to about 6 atoms. An exemplary but not exhaustive listing ofspecific moieties within these preferences is given below (with thegroup Y also shown to indicate the attachment position):

[0082] In the preceding compounds, where an amino group is separatedfrom Y by one or more methylene (CH₂) groups, higher or lower homologsmay be used, provided the separation between Y and the amino group iskept between about 2 and 6 atoms.

[0083] In other embodiments, Y is NR₃ in which R₃ is an alkyl orheteroalkyl group, or is optionally linked to R₂ to form a cyclicstructure. The ring thus formed can contain additional heteroatommoieties, such as —NH—, —NMe-, —O—, —N(lower alkyl)-, and the like aspart thereof and may be substituted or unsubstituted, as illustratedbelow:

[0084] Generally, R₂ will bear a polar group. For those embodiments inwhich Y is NR₃, the presence of one polar group (Y) will reduce the needfor additional polar groups on R₂. In such instances, R₂ may be equal toR₃—that is the partial formula R₂(CR₁)₂Y reduces to R₃(CR₁)₂NR₃, wherethe two R₃'s are independently variable.

[0085] Turning now to a series of particularly preferred compoundsrepresented by the formula (Ia), repeated below for convenience,specific and/or preferred embodiments relating thereto are now discussedin detail.

[0086] In formula Ia, Each Q is independently selected from —(CH₂)₂—,(CH₂)₃—, and a heteroaromatic ring independently selected fromsubstituted or unsubstituted imidazole, pyrrole, pyrazole, furan,isothiazole, oxazole, isoxazole, thiazole, furazan, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole,1,2,4-triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, and thiophene rings.Preferably, Q is a thiophene ring. Exemplary substituents include Cl, F,CH₃ (e.g., as in N-methylpyrrole or N-methylimidazole), and hydroxy(e.g., as in 3-hydroxypyrrole).

[0087] The subscripts a, b, and d are each independently 0, 1, 2, 3, 4,or 5, with the proviso that at least one of a, b, or d is other than 0.The subscripts c, e and f are each independently 0 or 1.

[0088] In the preferred embodiments of formula Ia, R₅ is selected fromhalogen, OR₇ and N(R₇)₂. More preferably, R₅ is selected from halogenand N(R₇)₂. R₆ is selected from hydrogen, halogen, a substituted orunsubstituted (C₁-C₁₂)alkyl group and a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group. When either of R₅ or R₆ is halogen, chlorineand fluorine are preferred, with chlorine being the most preferred. EachR₇ is independently selected from hydrogen, a substituted orunsubstituted (C₁-C₁₂)alkyl group and a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group. The alkyl groups in R₆ and R₇ can be, forexample, methyl, ethyl, propyl, isopropyl, butyl, s-butyl, isobutyl,t-butyl, pentyl, cyclopentyl, benzyl, or cyclohexyl, with methyl beingpreferred. In some embodiments, the (C₁-C₁₂)alkyl and heteroalkyl groupsare substituted with functional groups such as alkene; alkyne; hydroxy;primary, secondary, or tertiary amine; quaternary ammonium; alkoxy;acyl; amide; thiol; thioether; sulfoxide; sulfonamide; halogen; acycloaliphatic group; a heterocyclic group; an aromatic group; aheteroaromatic group; and the like; and combinations thereof.

[0089] In N(R₇)₂, one preferred embodiment is each R₇ is hydrogen. Inanother preferred embodiment, one R₇ is hydrogen and the other R₇ ismethyl.

[0090] Where R₅ is N(R₇)₂, the two R₇'s optionally can be joined to forma substituted or unsubstituted 4, 5, 6, or 7 membered ring optionallycontaining an —NH—, —NMe-, —O—, or N-lower alkyl group as part of thering. Exemplary embodiments of these compounds are shown by the partialformulae-below:

[0091] The 4, 5, 6, or 7 member ring formed by N(R₇)₂ may substituted orunsubstituted. In particular, the substituent may be or may contain anamino group.

[0092] Specific preferred combinations of R₂, R₁, and Y to form thepartial structure

[0093] in formula (I), above, and specific R₅'s in formula (Ia), above,are given below:

[0094] wherein r is 2, 3, or 4 and s is 1, 2, 3, 4, 5, or 6.

[0095] In view of the foregoing, below are disclosed preferred compoundswithin the scope of this invention:

[0096] In formula V, R₅ and R₇ have the meanings previously provided forformula Ia; m is 2, 3, or 4 and n is 0 or 1. At least one of R₅ and R₇is a positively charged group. Compound m n

V-a 3 0

V-b 3 0

V-c 3 0

V-d 3 0

V-e 3 0

V-f 3 0

V-g 3 0

V-h 3 0

V-i 3 0

V-j 3 0

V-k 3 0

V-l 3 0

V-m 3 0

V-n 3 0

V-o 3 0

V-p 3 0

V-q 3 0

V-r 2 1

V-s 2 1

V-t 4 1

V-u 3 1

V-v 3 0

V-w 3 0

V-x 3 0

V-aa 3 0

V-bb 2 0

V-cc 2 1

V-dd 3 0

V-ee 3 0

V-ff 3 0

V-gg 3 0

V-hh 3 0

V-ii 3 0

V-jj 3 0

V-kk 3 0

V-ll 3 0

V-mm 3 0

V-nn 3 0

V-oo 3 0

V-pp 3 0

V-qq 3 0

V-rr 3 0

V-ss 3 0

V-tt 3 0

V-uu 3 0

[0097]

[0098] In formula VI, b is 1, 2, 3, or 4, f is 0 or 1, and R₅ and R₇ areas previously defined. At least one of R₅ and R₇ contains a positivelycharged group. Compound b f

VI-a 3 1

VI-b 3 1

VI-c 2 1

[0099]

[0100] The structures of foregoing compounds were confirmed by at leastone of ¹H-NMR and mass spectrometry. In most instances, both ¹H-NMR andmass spectra were obtained.

[0101] Pharmaceutically acceptable salts of compounds of this inventioninclude salts of their conjugate acids or bases. Exemplary suitablecounterions for conjugate acid salts include the chlorides, bromides,phosphates, sulfates, maleates, malonates, salicylates, fumarates,ascorbates, methanesulfonates, malates, citrates, acetates, tartrates,succinates, glutamates, and the like, in particularly those salts whichare FDA acceptable. A conjugate acid salt may be formed by contacting acompound in the free base form with a sufficient amount of the desiredacid. A conjugate acid or base form of a compound of this invention isconsidered equivalent to the free base form (or the free acid form, asthe case may be) for the purposes of the claims of this invention.

[0102] Compounds of this invention are useful because they are strongDNA binders, often as nanobinders (i.e., association constant (K_(a)) of10⁹ M⁻¹) or even as picobinders (K_(a) of 10¹² M⁻¹). It is especiallynoteworthy that some compounds of the invention are nanobinders whilehaving relatively few heteroaromatic moieties (3-5), while previouslydescribed nanobinders have generally required a larger number ofheteroaromatic moieties.

[0103] Additionally, compounds of this invention have been found to haveanti-fungal (e.g., yeast, filamentous fungi) and/or anti-bacterial(gram-positive, gram-negative, aerobic, anaerobic) properties andtherefore may be used for combating (i.e., preventing and/or treating)infections by such pathogens. Other pathogens against which compounds ofthis invention may be used include protozoa and viruses. For humananti-infective applications, a compound of this invention may be used incombination with a pharmaceutically acceptable carrier. The compositionmay be dry, or it may be a solution. Treatment may be reactive, forcombating an existing infection, or prophylactic, for preventinginfection in an organism susceptible to infection.

[0104] Host organisms that may be treated include eukaryotic organisms,in particular plants and animals. The plant may be an agriculturallyimportant crop, such as wheat, rice, corn, soybean, sorghum, andalfalfa. Animals of interest include mammals such as bovines, canine,equines, felines, ovines, porcines, and primates (including humans).

[0105] While not wishing to be bound by any particular theory, it isbelieved that the compounds of this invention derive their biologicalactivity by binding to double stranded nucleic acid, in particulardouble stranded DNA.

[0106] The matching of a compound of this invention against a particularpathogen may be accomplished by rational design if the desired targetdsDNA base pair sequence—e.g., a sequence in a gene (or a regulatoryregion thereof, e.g., a promoter, enhancer) that is critical toproliferation of the pathogen—is known. In such circumstances, a nucleicacid binding moiety that binds to the target base pair sequence with thedesired degree of specificity is preferably used. The NABM may be aresidue of a naturally occurring dsDNA binder with known specificity forthe target sequence, or may be a synthetic dsDNA binder synthesizedaccording to the base pair recognition rules discussed hereinabove.Alternatively, the matching may be accomplished by a screening method,comprising the steps of (a) providing a compound of this invention to apopulation of pathogenic organisms and (b) determining whether thecompound inhibits proliferation of the population of pathogenicorganisms. A specific target pathogen may be screened against a libraryof compounds to determine which one(s) are effective against it.Conversely, a specific compound may be screened against a number ofpathogens, to determine which one(s) it is effective against.

[0107] The practice of this invention may be further understood byreference to the following examples, which are provided by way ofillustration and not of limitation.

[0108] Synthesis

[0109] A solid-phase and a solution-phase method are each described withreference to particular compounds. However, those skilled in the artwill understand these methods are general in nature and that othercompounds of this invention can be synthesized by variations in theprovided descriptions, with the appropriate substitution of startingmaterials, intermediates, and/or reagents.

[0110] Exemplary Solid-Phase Synthesis

[0111] The solid phase synthesis of compound V-s is described in thissection, as exemplary.

[0112] The starting point is commercially availableBoc-β-alanine-PAM-resin (see also Dervan et al., U.S. Pat. No. 6,090,947(2000)). This resin has a Boc-protected β-alanyl residue attached topolystyrene resin via a phenylacetamidomethyl (PAM) linkage:

[0113] Boc-β-alanine-PAM resin

[0114] Several “shorthand” notations will be used hereinafter, to makethe formulas more compact. “β” denotes the β-alanyl residue

[0115] Boc refers to the butyloxycarbonyl protective group (t-BuOC═O).“Py” denotes the residue

[0116] To illustrate, Boc-β-alanine-PAM resin may be represented as

[0117] the formula

[0118] represents compound V-s, and Boc-Py₂-OH represents

[0119] Scheme A below summarizes the synthetic route to compound V-s(shown in the scheme as A-4):

[0120] Coupling of Boc-Py₂OH to Boc-β-PAM resin. 1.25 gramsBoc-β-alanine-PAM resin (0.88 mmole/gram, A-1 in Scheme A) was treatedwith 30 mL trifluoroacetic acid (TFA) for 1 hour. The resin was airdried after washing with chloroform, methanol, and diethyl ether.

[0121] 0.48 grams BocPy₂OH (1.3 mmole, 1.2 eq) was activated with 0.49grams of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (“HBTU,” 1.3 mmole, 1.2 eq) in 2 mL DMF and 1 mLtriethylamine (TEA) for 10 min at 37° C. The resulting solution wasadded to the dried resin, followed by enough DMF to make a slurry, andput at 37° C. for 3 hours with shaking. 1 mL of acetic anhydride wasadded and the resin treated at room temperature for 30 min, to yieldBoc-Py₂-O—PAM resin (A-2 in Scheme A).

[0122] Deprotection of Boc-Py₂-β-PAM resin and coupling to4,5-dichloroisothiazole-3-carboxylic acid. The resin was filtered,washed with DMF, washed with chloroform, then treated with 100 mL TFAfor 1 hour and then dried as before, to yield the deprotectedH-Py₂-β-PAM resin.

[0123] 4,5-Dichloroisothiazole-3-carboxylic acid (0.26 grams, 1.3 mmole,1.2 eq) was activated with 0.48 grams HBTU (1.3 mmole, 1.2 eq) in 2 mLDMF and 1 mL triethylamine (TEA) for 5 min at room temperature. Theresulting solution was added to the dried resin, followed by enough DMFto make a slurry, and put at 37° C. for 3 hours with shaking to yieldthe resin-coupled isothiazole intermediate shown as A-3 in Scheme A.

[0124] Reaction with 3-(dimethylamino)propylamine—decoupling from resinwith simultaneous substitution in isothiazole ring. To produce compoundV-s and decouple it from the solid-phase support, the intermediate A-3was treated with 3 mL 3-(dimethylamino)propylamine (“Dp”) at 55° C. for12 hours. The solution was then filtered, washed with glacial aceticacid, diluted to 14 mL with glacial acetic acid, and purified bypreparative HPLC to yield compound V-s (shown as A-4 in Scheme A)

[0125] Reaction with 3-(dimethylamino)propylamine—decoupling from resinwithout substitution in isothiazole ring. Treatment with Dp under milderconditions results in decoupling only, substantially withoutsubstitution at one of the chlorines in the isothiazole group. Thus,treatment with 1.5 mL DMF and 1.5 mL Dp at 37° C. for 12 hours, followedby filtration, washing with glacial acetic acid, dilution to 14 mL withglacial acetic acid, and purification by preparative HPLC yielded thedichloro compound V-cc (shown as A-5 in Scheme A).

[0126] Preparative HPLC was performed under the following conditions:Hamilton PRP-1 reversed phase column, 250 mm×21.5 mm; solvent A: 0.5%acetic acid; solvent B: acetonitrile; 0-60% B in 180 min.

[0127] Pure fractions were lyophilized to provide 5-30 mg product.

[0128] Compound V-cc (and its analogs) is not only a useful precursorfor the synthesis of other compounds according to this invention bysubstitution of one of the isothiazole chlorines, but in its own rightwas a good binder for dsDNA and had significant anti-pathogenicproperties.

[0129] Exemplary Solution-Phase Synthesis

[0130] This example describes illustratively the solution synthesis ofcompounds such as V-a, V-b, and V-c. First, Scheme B below describes thesynthesis of intermediate compound B-8 (or H-Py₃-OH per the abovedescribed shorthand notation).

[0131] 4-nitro-2-trichloroacetyl-1-methylpyrrole (B-1). To a wellstirred solution of trichloroacetyl chloride (1 kg, 5.5 mole) in 1.5liter ethyl ether in a 12 liter flask was added dropwise over a periodof 3 h a solution of N-methylpyrrole (0.45 kg, 5.5 mole) in 1.5 literanhydrous ethyl ether. The reaction was stirred for an additional 3hours and quenched by the dropwise addition of a solution of 400 gpotassium carbonate in 1.5 liters water. The layers were separated andthe ether layer concentrated in vacuo to provide2-(trichloroacetyl)pyrrole (1.2 kg, 5.1 mol) as a yellow crystallinesolid sufficiently pure to be used without further purification. To acooled (−40° C.) solution of 2-(trichloroacetyl) pyrrole (1.2 kg, 5.1mol) in acetic anhydride (6 L) in a 12 L flask equipped with amechanical stirrer was added 440 mL fuming nitric acid over a period of1 hour while maintaining a temperature of (−40° C.). The reaction wascarefully allowed to warm to room temperature and stir an additional 4h. The mixture was cooled to −30 ° C., and isopropyl alcohol (6 L)added. The solution was stirred at −20° C. for 30 min during which timea white precipitate forms. The solution was allowed to stand for 15 minand the resulting precipitate collected by vacuum filtration to provide4-nitro-2-trichloroacetyl-1-methylpyrrole, shown as compound B-1 inScheme B (0.8 kg, 54% yield) TLC (7:2 benzene/ethyl acetate) Rf 0.7; ¹HNMR (DMSO-d₆) δ8.55 (d, 1 H, J=1.7 Hz), 7.77 (d, 1 H, J=1.7 Hz), 3.98(s, 3 H); ¹³C NMR (DMSO-d₆) δ173.3, 134.7, 133.2, 121.1,116.9,95.0,51.5; IR(KBr) 1694, 1516, 1423, 1314, 1183, 1113, 998, 750.FABMS m/e 269.936 (M+H 269.937 calc. for C₇H₅N₂O₃Cl₃).

[0132] Methyl 4-nitropyrrole-2-carboxylate (B-2). To a solution ofcompound B-1 (800 g, 2.9 mol) in 2.5 L methanol in a 4 L Erlenmeyerflask equipped with a mechanical stirrer was added dropwise a solutionof NaH (60% dispersion in oil) (log, 0.25 mol) in 500 mL methanol. Thereaction was stirred 2 h. at room temperature, and quenched by theaddition of conc. sulfuric acid (25 mL). The reaction was then heated toreflux, allowed to slowly cool to room temperature as Methyl4-nitropyrrole-2-carboxylate (shown as compound B-2 in Scheme B)crystallizes as white needles which were collected by vacuum filtrationand dried in vacuo. (450 g, 47% yield). TLC (ethyl acetate) Rf 0.8; ¹HNMR (DMSO-d₆) δ8.22 (d, 1 H, J=1.7 Hz), 7.22 (d, 1 H, J=1.6 Hz), 3.88(s, 3 H), 3.75 (s, 3 H);

[0133]¹³C NMR (DMSO-d₆) δ37.8, 52.2, 112.0, 123.0, 129.9, 134.6, 160.3;IR (KBr) 3148, 1718, 1541, 1425, 1317, 1226, 1195, 1116, 753. FABMS m/e184.048 (M+H 184.048 calc. for C₇H₈N₂O₄).

[0134] Methyl 4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride(B-3). Compound B-2 (450 g, 2.8 mol) was dissolved in ethyl acetate (8L). A slurry of 40 g of 10% Pd/C in 800 mL ethyl acetate was then addedand the mixture stirred under a slight positive pressure of hydrogen(c.a. 1.1 atm) for 48 h. Pd/C was removed by filtration through Celite,washed 1×50 mL ethyl acetate, and the volume of the mixture reduced toc.a. 500 mL. 7 L of cold ethyl ether was added and HCl gas gentlybubbled through the mixture. The precipitated amine hydrochloride wasthen collected by vacuum filtration to yield (380 g, 81.6%) of methyl4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride (shown as compoundB-3 in Scheme B) as a white powder. TLC (ethyl acetate) Rf(amine) 0.6,Rf salt (0.0), ¹H NMR (DMSO-d₆) δ10.23 (br s, 3H), 7.24 (d, 1H J=1.9),6.79 (d, 1H, J=2.0), 3.83 (s, 3H), 3.72 (s, 3H) ¹³C NMR (DMSO-d₆)δ160.8, 124.3, 121.2, 113.4, 112.0, 51.8, 37.1; IR (KBr) 3095, 2693,1709, 1548, 1448, 1266, 1102, 802, 751. FABMS m/e 154.075 (154.074 calc.for C₇H₁₀N₂O₂).

[0135] 4-[(tert-butoxycarbonyl)aminol-]1-methylpyrrole-2-carboxylic acid(B-4). Compound B-3 (340 g, 1.8 mol) was dissolved in 1 L of 10% aqueoussodium carbonate in a 3 L flask equipped with a mechanical stirrer,di-t-butyldicarbonate (400 g, 2.0 mmol) slurried in 500 mL of dioxanewas added over a period of thirty min maintaining a temperature of 20°C. The reaction was allowed to proceed for three h and was determinedcomplete by TLC, cooled to 5° C. for 2 h and the resulting whiteprecipitate collected by vacuum filtration. The Boc-pyrrole estercontaminated with Boc-anhydride was dissolved in 700 mL MeOH, 700 mL of2M NaOH was added and the solution heated at 60° C. for 6 h. Thereaction was cooled to room temperature, washed with ethyl ether (4×1000mL), the pH of the aqueous layer reduced to c.a. 3 with 10% (v/v) H₂SO₄,and extracted with ethyl acetate (4×2000 mL). The combined ethyl acetateextracts were dried (sodium sulfate) and concentrated in vacuo toprovide a tan foam. The foam was dissolved in 500 mL of DCM and 2 Lpetroleum ether added, the resulting slurry was concentrated in vacuo.The reaction was redissolved and concentrated three additional times toprovide (320 g, 78% yield) of4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid (shownas compound B-4 in Scheme B) as a fine white powder. TLC (7:2benzene/ethyl acetate v/v) Rf (ester) 0.8, Rf (acid) 0.1. (ethylacetate), Rf (acid) 0.6, ¹H NMR (DMSO-d₆) δ12.10 (s, 11H), 9.05 (s, 1H),7.02 (s, 1H), 6.55 (s, 1H), 3.75 (s, 3H), 1.41 (s, 9H) ¹³C NMR (DMSO-d₆)δ162.4, 153.2, 123.3, 120.1, 119.2, 107.9, 78.9, 36.6, 28.7.; IR(KBr)3350, 2978, 1700, 1670, 1586, 1458, 1368, 1247, 1112, 887, 779. FABMSm/e 241.119 (M+H241.119calc. for C₁₁H₁₇N₂O₄).

[0136]4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylpyrrole)-2-carboxylicacid (B-5). To a solution of compound B-4 (40 g, 167 mmol) in 150 mL DMFwas added 1.2 eq HOBt (27 g, 0.2 mmol) followed by 1.2 eq DCC (40.4 g,0.2 mmol). The solution was stirred for 5 h, and the DCU removed byfiltration, and rinsed with 50 mL of DMF. Compound B-3 (34 g, 160 mmol)was added, followed by TEA (80 mL) and the reaction stirred at 50° C.for 10 h. The reaction mixture was then added dropwise to a stirredsolution of ice water (2 L) and the solution placed at 4° C. overnight.The resulting precipitate was collected by vacuum filtration and driedovernight to provide ethyl4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylimidazole)-2-carboxylate (53 g, 83% yield). The ester was dissolved in200 mL methanol and 3M NaOH (200 mL) added and the resulting mixturestirred for 3 h at 50° C. Excess methanol was removed in vacuo and theresulting solution acidified by the addition of 2 M HCl. The resultingprecipitate of4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylpyrrole)-2-carboxylicacid (shown as compound B-5 in Scheme B) was collected by vacuumfiltration and dried in vacuo to yield a white powder. (43 g, 90%yield).

[0137]4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylpyrrole)-2-(4-carboxamido-1-methylpyrrole)-2-carboxylicacid (B-6). 150 mL of DMF, followed by 80 mL of TEA was added to amixture of compound B-5 acid (50 g, 139 mmol) and 0.98 eq HBTU (51 g).The solution was stirred for 2 h at rt. 1.1 eq of compound B-3 (28.9 g)was added, and the reaction stirred at 50° C. for 30 h. The reactionmixture was then added dropwise to a stirred solution of ice water (1.2L) and the solution the resulting precipitate was collected by vacuumfiltration and dried overnight to provide the methyl ester of compoundB-6 (59 g, 85% yield). The ester was dissolved in 200 mL methanol and2.5M NaOH (200 mL) added and the resulting mixture stirred for 10 h at50° C. Excess methanol was removed in vacuo and the resulting solutionacidified by the addition of 2 M H2SO4. The resulting precipitate of4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylpyrrole)-2-(4-carboxamido-1-methylpyrrole)-2-carboxylic(shown as compound B-6 in Scheme B) was collected by vacuum filtrationand dried in vacuo to yield a white powder. (51 g, 87% yield).

[0138] Reaction of Compound B-6 with 3-(dimethylamino)propylamine.Compound B-6 was converted to the corresponding amide with3-(dimethylamino)propylamine (compound B-7 in Scheme B) by the followingprocedure: Compound B-6 (46.8 grams, 0.1 mmole, 1 eq) was activated withHBTU (34.4 grams, 0.095 mmole, 0.95 eq) in 50 mL DMF and 25 mL TEA for45 min at room temperature. 3-(Dimethylamino)propylamine (12 mL, 0.12mmole, 1.2 eq) was added and the reaction was stirred at 37° C.overnight. The product mixture containing compound B-7 was concentratedin vacuo.

[0139] Deprotection of Compound B-7. To approximately 30 grams compoundB-7 was added 150 mL trifluoroacetic acid. The reaction was stirredovernight at room temperature. The product compound B-8 was concentratedin vacuo and approximately 40 mL acetic acid and 200 mL water was added(enough acetic acid to prevent precipitation) and the solution wasextracted with diethyl ether three times. Compound B-8 was purified bypreparative HPLC (Hamilton PRP-1 reversed phase column, 250 mm×101.6 mm;solvent A: 0.5% acetic acid, solvent B: acetonitrile; 0-100% B in 180min).

[0140] Coupling of Compound B-8 with4,5-dichloroisothiazole-3-carboxylic acid. 2 grams4,5-dichloroisothiazole-3-carboxylic acid (10 mmole, 1.2 eq) wasactivated with 3.7 grams HBTU (9.8 mmole, 1.14 eq) in 20 mL DMF and 10mL triethylamine. The solution was stirred for 10 minutes at roomtemperature. Compound B-8 (4 grams, 8.5 mmole, 1 eq) was added as asolid, and 4 mL DMF added to complete the transfer. The resultingsolution was stirred at 37° C. overnight. The reaction was then dried invacuo, 200 mL 10% aqueous acetic acid was added and the product waspurified by preparative HPLC. (Hamilton PRP-1 reversed phase column, 250mm×101.6 mm; solvent A: 0.5% acetic acid; solvent B: acetonitrile; 0-60%B in 320 min). Pure fractions were lyophilized to provide 2 gramsproduct compound V-v (3.1 mmole, 36% yield). Compound V-v can be usedfor the synthesis of a variety of compounds by substitution of the C-5chlorine with an appropriate amine, alkoxide, or thiolate. In the caseof reaction with a thiolate, the resulting thioether may be oxidized tothe corresponding sulfone or sulfoxide. Further, the compound may havealkylating activity.

[0141] Aminolysis of Compound V-v. The following procedure generallydescribes the aminolysis of Compound V-v to yield compounds such as V-a,V-b, or V-c. 20 mg of Compound XII was dissolved in 1 mL of neat amine(or 1 gram of amine with 1 mL DMF if the amine is a solid at roomtemperature). The amines are 4-(dimethylamino)butylamine,3-(dimethylamino)propylamine, and 2-(dimethylamino)ethylamine for thesyntheses of compounds V-a, V-b, and V-c, respectively. The reaction washeated at 55° C. for 15 hours, then cool to room temperature. Aceticacid was added to a total volume of 14 mL and the product mixture wasloaded onto a preparative HPLC column (Hamilton PRP-1 reversed phasecolumn, 250 mm×21.5 mm). Solvent A: 0.5% aqueous acetic acid; solvent B:acetonitrile; 0-60% solvent B in 180 min.

[0142] The compound structures of this invention were confirmed by massspectroscopy or ¹H-NMR spectroscopy, or in most instances, both. Thespectra were in each instance consistent with the assigned structures.

[0143] Binding to dsDNA

[0144] Quantitative DNase I footprint titration experiments of polyamideNABMs indicate that the isothiazole group has a preference for bindingopposite G relative to T, A, and C. While not excluding othermechanisms, it is likely that the N2 nitrogen of the isothiazole ringmakes a specific hydrogen bond with the N2 exocyclic amine group of G.The DNA-binding affinities of the isothiazole containing polyamidesreveal that this interaction is likely energetically favored relative tothe comparable interaction of the N-methylimidazole-2-carboxamide groupwith G. Surprisingly, the affinities of a number of polyamidescontaining the isothiazole heterocycle and not more than a total of fourheterocycle rings are greater than about 10⁹ M⁻¹. Polyamides containingthe isothiazole group can bind to DNA via a variety of binding motifsincluding but not limited to side-by-side overlapped “2:1”polyamide-DNA-binding, side-by-side slipped 2:1 polyamide-DNA-binding,hairpin polyamide-DNA-binding, and 1:1 polyamide-DNA-binding. For 2:1polyamide-DNA binding, the isothiazole group is preferably paired with a5-aminopyrrole-2-carboxylic acid residue or its analogs or β-alanineresidue or its analogs. Binding studies further indicate that polyamidescontaining the isothiazole group can bind DNA with both a 5′-3′N-terminus-to-C-terminus (N—C), DNA-strand—polyamide orientation as wellas a 5′-3′ C—N DNA strand—polyamide orientation. Molecules that presenta positively charged moiety on both the C-terminal and N-terminal endsmost likely bind the 5′-3′ C—N orientation preferentially.

[0145] A partial nucleotide sequence of a dsDNA restriction fragmentused for footprinting experiments is:5′-CTAGATGCCGCTAAGTACTATGCCGCTAACTACT (SEQ ID NO:1) ATGCCGCTAATTACTATGCCGCTAAATACTATGCCG CTAACTAGTATGCCGCTATGCA-3′.

[0146] Other DNA molecules having a nucleotide sequence to be targetedby a NABM can be readily synthesized according to well known methods.

[0147] The binding data for representative compounds appears below inTable A TABLE A dsDNA Binding Results Sequence, Binding Constant (Mi)Compound AGTACT ACTACT ATTACT AATACT TGGTCA V-u  1 × 10¹¹ 1 × 10⁹ 5 ×10⁸ 1 × 10⁸ — v-t 2 × 10⁸ 2 × 10⁸ 2 × 10⁸ 2 × 10⁸  1 × 10¹⁰ V-c 5 × 10⁹2 × 10⁸ 2 × 10⁸ 2 × 10⁸ — V-b 3 × 10⁹ 2 × 10⁸ 1 × 10⁸ 1 × 10⁸ — V-a 3 ×10⁹ 1 × 10⁸ 1 × 10⁸ 1 × 10⁸ — VI-a  2 × 10¹⁰ 2 × 10⁹ 2 × 10⁹ 2 × 10⁹ —VI-b  1 × 10¹¹ 7 × 10⁹ 7 × 10⁹ 7 × 10⁹ — VI-c 2 × 10⁸ 1 × 10⁸  1 × 10¹⁰1 × 10⁹ — XII (comparative) 3 × 10⁹ 5 × 10⁸ 5 × 10⁸ 5 × 10⁸ — (XII)

[0148] Compounds V-u, V-c, V-b, and V-a were screened on a restrictionfragment containing over 300 different 6-bp sites. Each of thesecompounds was found to bind preferentially to an AGTACT site withsubnanomolar affinities ranging from 1×10¹¹ M⁻¹ to 3×10⁹ M⁻¹. It isnotable that these affinities are significantly higher than expected forcompounds containing 4 heterocycles. Specificity against the single basepair mismatch sites ACTACT, ATTACT, and AATACT was determined to rangefrom 200-fold to 11-fold for each of these compounds. Compound V-t wasfound to bind a TGGTCA site with an affinity of 1×10¹⁰M⁻¹ preferentiallyto AGTACT, ACTACT, ATTACT, or AATACT. The mechanism of this binding isnot certain, but may involve either a “slipped” 2:1 binding mode (see,e.g., U.S. Pat. No. 6,090,947) or a 1:1 binding mode. Compounds VI-a andVI-b most likely adopt hairpin conformations. Each of these compoundswas found to bind preferentially to an AGTACT site with subnanomolaraffinities of 2×10¹⁰ M⁻¹ and 1×10¹¹ M⁻¹ respectively. Each of theseDNA-binding affinities is 9-30 fold higher than that of the comparisoncompound XII, having an isothiazole ring replaced by an imidazole ring.Compound VI-c was observed to bind ATTACT, and AATACT preferentially toACTACT and AGTACT, potentially as a single base mismatch. The optimaltarget sequence for this compound may be WGWCWW (W=A or T).

[0149] Exemplary protocol for DNaseI footprint titration experiments.All reactions were executed in a total volume of 400 μL. A polyamidestock solution or H₂O (for reference lanes) was added to an assay buffercontaining 3′-³²P radiolabeled restriction fragment (20,000 cpm),affording final solution conditions of 10 mM Tris.HCl, 10 mM KCl, 10 mMMgCl₂, 5 mM CaCl₂, pH 7.0. The solutions were allowed to equilibrate forat least 12 hours at 22° C. Footprinting reactions were initiated by theaddition of 10 μL of a stock solution of DNase I (at the appropriateconcentration to give 55% intact DNA) containing 1 mM dithiothreitol andallowed to proceed for 7 minutes at 22° C. The reactions were stopped bythe addition of 50 μL of a solution containing 2.25 M NaCl, 150 mM EDTA,23 μM base pair calf thymus DNA, and 0.6 mg/ml glycogen, and ethanolprecipitated. The reactions were resuspended in 1×TBE/80% formamideloading buffer, denatured by heating at 85° C. for 15 minutes, andplaced on ice. The reaction products were separated by electrophoresison an 8% polyacrylamide gel (5% crosslinking, 7 M urea) in 1×TBE at 2000V for 1.5 h. Gels were dried on a slab dryer and then exposed to astorage phosphor screen at 22° C.

[0150] Quantitative DNase I Footprint Titration Data Analysis.Background-corrected volume integration of rectangles encompassing thefootprint sites and a reference site at which DNase I reactivity wasinvariant across the titration generated values for the site intensities(I_(site)) and the reference intensity (I_(ref)). The apparentfractional occupancy (θ_(app)) of the sites were calculated using theequation: $\begin{matrix}{\theta_{app} = {1 - \frac{I_{site}/I_{ref}}{I_{site}{{^\circ}/I_{ref}}{^\circ}}}} & (1)\end{matrix}$

[0151] where I_(site)° and I_(ref)° are the site and referenceintensities, respectively, from a DNase I control lane to which nopolyamide was added.

[0152] The ([L]_(tot), θ_(app)) data were fit to a Langmuir bindingisotherm (eq. 2, n=1) by minimizing the difference between θ_(app) andθ_(fit), using the modified Hill equation: $\begin{matrix}{\theta_{fit} = {\theta_{\min} + {\left( {\theta_{\max} - \theta_{\min}} \right)\frac{{K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}{1 + {K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}}}} & (2)\end{matrix}$

[0153] where [L_(tot)] is the total polyamide concentration, K_(a) isthe equilibrium association constant, and θ_(min) and θ_(max) are theexperimentally determined site saturation values when the site isunoccupied or saturated, respectively. The data were fit using anonlinear least-squares fitting procedure of KaleidaGraph software (v.3.0.1, Abelbeck Software) with K_(a), θ_(max), and θ_(min) as theadjustable parameters. The goodness of fit of the binding curve to thedata points is evaluated by the correlation coefficient, with R>0.97 asthe criterion for an acceptable fit. All lanes from a gel were usedunless a visual inspection revealed a data point to be obviously flawedrelative to neighboring points. The data were normalized using thefollowing equation: $\begin{matrix}{\theta_{norm} = \frac{\theta_{app} - \theta_{\min}}{\theta_{\max} - \theta_{\min}}} & (3)\end{matrix}$

[0154] Biological Activity

[0155] Compounds of this invention were screened for their in vitroactivities against different species of bacteria and fungi. Theirminimal inhibition concentration (MIC) was determined using the NationalCommittee for Clinical Laboratory Standards (NCCLS) broth microdilutionassay in microtiter plates, as set forth in: (1) the guidelines of theNational Committee for Clinical Laboratory Standards (NCCLS) DocumentM7-A4 (NCCLS, 1997); (2) the guidelines of the National Committee forClinical Laboratory Standards (NCCLS) Document M11-A4 (NCCLS, 1997); and(3) the guidelines and reference method of the National Committee forClinical Laboratory Standards (NCCLS) Document M27-T (NCCLS, 1995). Forantifungal essays, the method in Murray, P R., 1995 Manual of clinicalmicrobiology (ASM Press, Washington, D.C.), was employed. A variety ofGram-positive and Gram-negative bacteria (aerobes and anaerobes) as wellas yeasts and filamentous fungi were tested. These organisms includedStaphylococcus spp., Streptococcus spp., Enterococcus spp.,Corynebacterium spp., Listeria spp., Bacillus spp., Micrococcus spp.,Peptostreptococcus spp, Clostridium spp., Propionibacterium spp.,Escherichia spp., Pseudomonas spp., Haemophilus spp., Candida spp.,Cryptococcus spp., Aspergillus spp., Trichophyto spp., Paecilomycesspp., Saccharomyces spp. and Fusarium spp. In addition, effectivenessagainst some drug resistant microbes were evaluated. Other pathogenicbacteria against which compounds of this invention may be effectiveinclude Acinetobacter spp., Alcaligenes spp., Campylobacter spp.,Citrobacter spp., Enterobacter spp., Proteus spp., Salmonella spp.,Shigella spp., Helicobacter spp., Neisseria spp., Vibrio spp.,Bacteroides spp., Prevotella spp., Mycoplasma spp., Mycobacteria spp.,and Clamydia spp.

[0156] Compounds such as V-a, V-b, V-c, V-r, V-s, V-q, V-w, V-x/V-y,V-z/V-aa, V-bb and VIII demonstrated excellent antimicrobial activitiesagainst numerous species (Tables I, la and II), especially againstGram-positive bacteria and fungi. The MIC's for these compounds werebetween ≦0.062 μg/mL to >128 μg/mL. One of the most potent compounds,V-a, has MIC's less than 8 μg/mL for most species tested, with theexception of Gram-negative bacteria such as Escherichia and Pseudomonas.The results indicated that these compounds were broad-spectrumantimicrobial agents. Table Ia and IIa, below, also provides comparativedata against the prior art antibiotics distamycin, netropsin, andofloxacin.

[0157] As shown in the preliminary screening, compounds of thisinvention were active against the isolates resistant to conventionalantibiotics, including methicillin resistant staphylococcus aureus,multiple drug resistant Streptococcus pneumoniae, vancomycin resistantEnterococcus faecium and polyene resistant Candida albicans.

[0158] The minimal bactericidal concentration (MBC) has been determinedfor some of these designed compounds according to NCCLS guideline(Lorian, V. 1996 Antibiotics in laboratory medicine (The Williams &Wilkins Co., Baltimore, Md.)). The difference between the MICs and theMBCs has been established as an index of the bactericidal activity of anantibiotic. The results shown in Table III show that compounds V-a andV-b were microbicidal agents for all species tested.

[0159] Compounds of this invention have broad spectrum anti-Grampositive bacteria and antifungal potency. Based on the pattern ofantimicrobial activity of these polyamides, the antimicrobial activityshould extend into other bacterial and fungal species not listed in theTable I or II. In addition, the compounds have the properties activeagainst the microbes resistant to conventional antibiotics andantifungal agents. These compounds and their analogs can be used inantimicrobial chemotherapeutics for the treatment of human or animalinfections as a systemic and/or topical agent.

[0160] In addition to the data presented in the tables below, compoundsV-w and V-bb were tested against Bacillus cereus, giving MIC's of 0.5and 8 μg/mL, respectively. TABLE I Antibacterial Activity Data Compound(MIC, μg/mL) Organism (ATCC) V-s V-a V-b V-c V-q V-r VIII Gram-positivebacteria (aerobes) Staphylococcus aureus (29213) >128 2 2 8 8-16 >128 4Staphylococcus aureus (33591) 64 2 4 16 16 ND 8 (methicillin resistant)Staphylococcus epidermidis >128 1 2 8 4 ND 8 (12228) Enterococcusfaecalis (29212) 8 1 1 4 1 16 1 Enterococcus faecium (51559) 128 1 2-4 84 ND 8 (vacomycin resistant) Streptococcus pneumoniae (49619) 16 0.5 0.52 2 16 1 Streptococcus pneunioniae (51422) ND 8 1 4 4 ND ND (multipledrug resistant) Streptococcus pyogenes (49339) 8 0.125 0.25 2 1 ND≦0.062 Listeria monocytogenes (19115) ND 4 8 ND ND ND 4 Bacillusanthracis ND 4 32 ND ND ND Bacillus subtilis (6633) 8 0.25 0.5-1 4 ND NDND Bacillus cereus (11778) 2-4 0.25 0.5 4 4-8 >128 ND Micrococcus luteus(381) 32 2 8 8 8 ND ND Corynebacterium Group A (49676) 2-4 0.25 0.5 1 1ND 1 Gram-positive bacteria (anaerobes) Propionibacterium acnes (33179)16 2 4 8 ND ND ND Clostridium perfringens (13124) 32- 1 1 2 ND ND ND 64Peptostreptococcus asaccharolytics 8 0.125 0.125 0.5 ND ND ND (29743)Gram-negative bacteria Haemophilus influenzae (49247) 64 32 32 NDND >128 ND Escherichia coli (25922) >128 >128 >128 >128 >128 >128 >128Pseudomona aeruginosa (27853) >128 >128 >128 >128 >128 ND ND

[0161] TABLE Ia Additional Antibacterial Activity Data Compound (MIC,μg/mL)* ORGANISM (ATCC) Distamycin Netropsin Ofloxacin V-x V-aaGram-positive bacteria (aerobes) Staphylococcus aureus (29213) 32 40.125 0.5 1 Staphylococcus aureus (33591) 16 4 0.25 1 2 (MRSA)Staphylococcus epidermidis 64 2-4 0.25 0.25 0.5-1 (12228) Streptococcuspneumoniae 16 0.125 1-2 0.125 0.25 (49619) Streptococcus Group A (49339)32 8 1 0.062 0.25 Enterococcus faecalis (29212) 16-32  2 8 0.25 1Enterococcus faecium (51559) 128 8 8 0.25 1 (VREF) Listeriamonocytogenes ND ND 1 0.25 0.5 (19115) Corynebacterium Group A 16 0.50.125 0.125 0.25 (49676) Micrococcus luteus (381) 32 ND 1 1 1 Bacillussubtilis (6633) 64-128 4 0.125 0.25 0.5 Bacillus cereus (11778) 8 20.125 0.25 0.5 Gram-positive bacteria (anaerobes) Propionibacteriumacnes 16 ND 0.25 ND ND (33179) Clostridium perfringens (13124) 8 ND 0.25ND ND Peptostreptococcus 8 ND 0.5 ND ND asaccharolytics (29743-1)Gram-negative bacteria Haemophilus influenzae ND 4 0.25 ND NDEscherichia coli (25922) >128 2 0.03 >128 >128 Pseudomona aeruginosa(27853) >128 >128 >128 ND ND

[0162] TABLE II Antifungal Activity Compound (MIC, μg/mL) Organism(ATCC) V-s V-a V-b V-c V-q V-r VIII Yeasts Candida albicans >128 4 4 8ND >128 ND (90028) Candida albicans ND 4 ND ND ND >128 ND (38247)(Polyene resistant) Candida tropicalis ND ND ND ND ND ND  2 (13803)Candida parasilosis ND ND ND ND ND    2 128 (10232) Cryptcoccus ND ND NDND ND    16  16 neoformans (90112) Saccharomyces ND 8 ND ND ND ND NDcerevisiae (44773) Filamentous fungi Aspergillus niger ND 2 4 ND ND NDND (10535) Fusarium solani ND 4 4-8 ND ND ND ND (36031) Paecilomyces ND0.5 0.5 ND ND ND ND variotii (22319) Trichophyto ND 16 16 ND ND ND NDtonsurans (28942)

[0163] TABLE IIa Additional Antifungal Activity Data ORGANISM Compound(MIC, μg/mL)* (ATCC) Distamycin Netropsin Ofloxacin V-x V-aa YeastsCandida albicans >128 ND ND ND ND (90028) Candida albicans >128 ND ND 416 (38247) Candida tropicalis >128 16 ND 2  4 (13803) Candida >128 16 ND2  4 parasilosis (10232) Cryptococcus >128  4 ND 2  4 neoformans (90112)

[0164] TABLE III Microbiocidal Activity Compound V-a (μg/mL) V-b (μg/mL)ORGANISM (ATCC) MIC MBC MIC MBC Staphylococcus aureus (29213) 2 2 2 4Staphylococcus aureus (33591) 2 4 4 ND (methicillin resistant)Staphylococcus epidermidis (12228) 1 1 2 ND Enterococcus faecalis(29212) 1 >4 1 >4 Enterococcus faecium (51559) 1 2-4 2-4 ND (vacomycinresistant) Streptococcus pneumoniae (49619) 0.5 0.5 0.5 0.5Streptococcus pyogenes (49339) 0.125 0.125 0.25 ND Bacillus subtilis(6633) 0.25-0.5 0.5 0.5-1 ND Bacillus cereus (11778) 0.25 0.5 0.5 0.5Micrococcus luteus (381) 0.25-0.5 0.5 0.5-1 ND Corynebacterium Group A(49676) 0.25-0.5 0.25 0.5 ND Propionibacterium acnes (33179) 4 8 4 NDClostridium perfringens (13124) 1 2 1 ND Peptostreptococcusasaccharolytics 0.125 0.25 0.125 ND (29743) Candida albicans (90028) 4 44 4 Saccharomyces cerevisiae (44773) 8 8 16 16

[0165] TABLE IV MIC, μg/mL S. aureus B. cereus E. coli C. albicansCompound (29213) (11778) (25922) (38247) V-x 0.5, 0.25, 32, 4, 1 2 128 8V-z 1 0.5 128 16 V-dd 1 1 128 4 V-ee 0.25, 0.25 64, 2 0.125 32 V-ff 1 232 16 V-gg 2, 2, 32 32 1 0.5 V-hh 1 1 32 32 V-ii 1 1 32 2 V-jj 0.5 0.2532 1 V-kk 0.5 0.25 32 0.25 V-ll 0.25 0.5 32 4 V-mm 0.5 0.25 32 32 V-nm0.031 0.064 32 1 V-oo 0.25 0.5 32 1 V-pp 0.5 0.5 32 4 V-qq 0.25 0.25 3232 V-rr 1 0.5 32 32 V-tt 0.5 0.25 32 32 V-uu 0.25 1 4, 32, 32 4

[0166] The foregoing detailed description of the invention includespassages that are chiefly or exclusively concerned with particular partsor aspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious descriptions herein relate to specific embodiments of theinvention, it is to be understood that where a specific feature isdisclosed in the context of a particular embodiment, such feature canalso be used, to the extent appropriate, in the context of anotherfigure or embodiment, in combination with another feature, or in theinvention in general.

[0167] Further, while the present invention has been particularlydescribed in terms of certain preferred embodiments, the invention isnot limited to such preferred embodiments. Rather, the scope of theinvention is defined by the appended claims.

What is claimed is:
 1. A charged compound represented by the formula (I)W—Y-[Het]-L-[NABM]  (I) and pharmaceutically acceptable salts thereof,wherein [NABM] is a double-stranded nucleic acid binding moiety; L isselected from the group consisting of a covalent bond and a linkinggroup; [Het] is a heteroaromatic moiety other than N-methyl orN-hydrogen pyrrole, selected from the group consisting of

wherein one of X₁, X₂, and X₃ is a ring vertex selected from the groupconsisting of —O—, —S—, and —NR₃—, and the other two of X₁, X₂, and X₃are ring vertices selected from the group consisting of ═N— and ═CR₄—; Yis O, S, S(O), SO₂, C(R₁)₂, N(R₃)SO₂, SO₂N(R₃), and NR₃; W is halogen ora group having the formula:

wherein each R₁ is independently selected from the group consisting ofH, F, and substituted or unsubstituted (C₁-C₆)alkyl; R₂ is a moietybearing a polar group if Y is other than NR₃ and is a moiety bearing apolar group, a substituted or unsubstituted (C₁-C₁₂)alkyl group or asubstituted or unsubstituted (C₁-C₁₂)heteroalkyl group if Y is NR₃; eachR₃ is independently selected from the group consisting of H, asubstituted or unsubstituted (C₁-C₁₂)alkyl group, and a substituted orunsubstituted (C₁-C₁₂)heteroalkyl group, provided that neither ofR₂(R₁)₂C and R₃ contains a 2-chloroethyl or 2-hydroxyethyl group when Yequals NR₃; and each R₄ is independently selected from the groupconsisting of hydrogen, a halogen, an amino group, a (C₁-C₈)alkylaminogroup, a di(C₁-C₈)alkylamino group, a tri(C₁-C₈)alkyl ammonium group, ahydroxyl group, a (C₁-C₈)alkoxy group, a thiol group, a (C₁-C₈)thioethergroup, a (C₁-C₈)sulfone group, a (C₁-C₈)sulfoxide group, a(C₁-C₈)sulfonamide group, a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group and a substituted or unsubstituted(C₁-C₁₂)alkyl group; provided that at least one of R₂, [Het], or [NABM]is positively charged under assay or physiological conditions.
 2. Acompound according to claim 1, wherein [NABM] is selected from the groupconsisting an interealator moiety, a minor groove binding moiety, and amajor groove binding moiety.
 3. A compound according to claim 1, wherein[NABM] binds in the minor groove of the double-stranded DNA.
 4. Acompound according to claim 1, wherein Y is NR₃.
 5. A compound accordingto claim 1, wherein each R₁ is hydrogen.
 6. A compound according toclaim 1, wherein R₃ is a substituted (C₁-C₁₂)alkyl group having at leastone substituent selected from the group consisting of a (C₁-C₄)alkenegroup, a (C₁-C₄)alkyne group, an amino group, a (C₁-C₈)alkylamino group,a di(C₁-C₈)alkylamino group, a tri(C₁-C₈)alkyl ammonium group, a hydroxygroup, a (C₁-C₈)alkoxy group, a thiol group, a (C₁-C₈)thioether group, a(C₁-C₈)sulfone group, a (C₁-C₈)sulfoxide group, a (C₁-C₈)sulfonamidegroup, a (C₁-C₈)acyl group, a mono or di(C₁-C₈) N-alkylamide group, athiol group, a (C₁-C₄)thioether group, a (C, -C₄)sulfone group, a(C₁-C₄)sulfoxide group, a mono or di(C₁-C₈) N-alkylsulfonamide group, ahalogen, a (C₃-C₇)cycloaliphatic group, a five-, six- or seven-memberedheterocyclic group, an aryl group, and a heteroaryl group.
 7. A compoundaccording to claim 1, wherein at least two of R₂, [Het], and [NABM] havea positive charge.
 8. A compound according to claim 1, wherein thepositive charge results from the protonation of a basic nitrogenselected from the group consisting of an amine nitrogen, an amidinenitrogen, a guanidine nitrogen, a pyridine nitrogen, a pyridazinenitrogen, a pyrazine nitrogen, a pyrimidine nitrogen, an imidazolenitrogen, and an aniline nitrogen.
 9. A compound according to claim 1,wherein the nucleic acid binding moiety [NABM] has at least one positivecharge-bearing moiety attached thereto as a pendant group or as aterminal group distal from [Het].
 10. A compound according to claim 9wherein the positive charge-bearing moiety is selected from the groupconsisting of a basic amino acid residue and a peptide unit comprisingat least one basic amino acid residue.
 11. A compound according to claim10, wherein the basic amino acid residue is selected from the groupconsisting of arginine, histidine, and lysine residues.
 12. A compoundaccording to claim 10, wherein the peptide unit comprises a prolineresidue separating two basic amino acid residues.
 13. A compoundaccording to claim 1, wherein the nucleic acid binding moiety does nothave a positive charge.
 14. A compound according to claim 1, wherein the[NABM] is selected from the group consisting of doxorubicin, daunomycin,anthramycin, calicheamycin, mitomycin, CC-1065, duocarmycin, distamycin,and netropsin, and analogs and derivatives thereof.
 15. A compoundaccording to claim 1, wherein the [NABM] is selected from the groupconsisting of pentamidine, berenil, stilbamidine, DDUG, NSC 101327, SN6999, SN 6136, SN 16814, SN18071, NSC 57153, Hoechst 33258, Ionen X,methyl green, and analogs and derivatives thereof.
 16. A compoundaccording to claim 1, wherein the nucleic acid binding moiety [NABM]comprises N-methylpyrrole carboxamide residues and optionally one ormore residues selected from the group consisting of N-methylimidazolecarboxamide residues, N-methyl-3-hydroxypyrrole carboxamide residues,β-alanine carboxamide residues, glycine carboxamide residues,5-aminovaleric acid carboxamide residues, γ-2,4-diaminobutyric acidcarboxamide residue, and y-aminobutyric acid carboxamide residues.
 17. Acompound according to claim 1, wherein the nucleic acid binding moiety[NABM] comprises a structure according to the formula (II):

-Q₁-Z₁-Q₂-Z₂- . . . -Q_(m)-Z_(m)-

  (II) wherein each of Q₁, Q₂, . . . , Q_(m) is independently selectedfrom the group consisting of a heteroaromatic moiety and (CH₂)_(p),wherein the subscript p is an integer from 1 to 3, inclusive; whereineach of Z₁, Z₂, . . . , Z_(m) is independently selected from the groupconsisting of a covalent bond and a linking group; and the subscript mis an integer of from 1 to
 9. 18. A compound according to claim 17,wherein at least one of Q₁, Q₂, . . . Q_(m) is a heteroaromatic moiety.19. A compound according to claim 18, wherein the heteroaromatic moietyis selected from the group consisting of substituted and unsubstitutedimidazole, pyrrole, pyrazole, furan, isothiazole, oxazole, isoxazole,thiazole, furazan, 1,2,3-thiadiazole, 1,2,4-thiadiazole,1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole,1,2,4-oxadiazole, 1,3,4-oxadiazole, and thiophene moieties.
 20. Acompound according to claim 17, wherein at least one of Z₁, Z₂, . . . ,Z_(m) is a linking group having 2, 3, 4, or 5 backbone atoms.
 21. Acompound according to claim 20, wherein each of Z₁, Z₂, . . . , Z_(m) isa carboxamide group.
 22. A compound according to claim 1, wherein thenucleic acid binding moiety [NABM] comprises a structure according tothe formula (III):

wherein each n is independently 1, 2, 3, 4, 5, 6, 7, 8, or 9, and each mis independently 0 or
 1. 23. A compound according to claim 22, whereineach n is 2 or 3 and each m is
 1. 24. A compound according to claim 1,wherein L comprises 2, 3, 4, or 5 backbone atoms.
 25. A compoundaccording to claim 24, wherein L is a carboxamide group.
 26. A compoundaccording to claim 1, wherein the heteroaromatic moiety [Het] isselected from the group consisting of substituted and unsubstitutedimidazole, pyrrole other than N-methyl or N-hydrogen pyrrole, pyrazole,furan, isothiazole, oxazole, isoxazole, thiazole, furazan,1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole,1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,4-oxadiazole,1,3,4-oxadiazole, and thiophene moieties.
 27. A compound according toclaim 1, wherein the heteroaromatic moiety [Het] is an isothiazolerepresented by a structure selected from the group consisting of:

wherein R₄ is as defined in claim
 1. 28. A compound according to claim1, represented by the formula (IVa):

wherein R₁, R₂, and R₄ are as defined in claim
 1. 29. A compoundaccording to claim 1, comprising a structure represented by formula(IVb):

wherein R₁, R₂, and R₄ are as defined in claim 1; each n isindependently 1, 2, 3, 4, 5, 6, 7, 8, or 9; and each m is independently0 or
 1. 30. A compound according to claim 1, wherein

is selected from the group consisting of

wherein r is 2, 3, or 4 and s is 1, 2, 3, 4, 5, or
 6. 31. A compound ofthe formula (Ia)

or a pharmaceutically acceptable salt thereof, wherein R₅ is a memberselected from the group consisting of halogen, OR₇ and N(R₇)₂; R₆ is a member selected from the group consisting of H, halogen, a substituted orunsubstituted (C₁-C₁₂)alkyl group and a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group; each R₇ is independently selected from thegroup consisting of H, a substituted or unsubstituted (C₁-C₁₂)alkylgroup and a substituted or unsubstituted (C₁-C₁₂)heteroalkyl group; eachQ is independently (CH₂)₂, (CH₂)₃, or a heteroaromatic group other thanan N-methylpyrrole group; each of a, b, and d is independently 0, 1, 2,3, 4, or 5, with the proviso that at least one of a, b, or d is otherthan 0; each of c, e and f is independently 0 or
 1. 32. A compoundaccording to claim 31, wherein a is 1 and Q is selected from the groupconsisting of substituted and unsubstituted imidazole, pyrrole,pyrazole, furan, isothiazole, oxazole, isoxazole, thiazole, furazan,1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole,1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,4-oxadiazole,1,3,4-oxadiazole, and thiophene moieties.
 33. A compound according toclaim 31, wherein Q is thiophene.
 34. A compound according to claim 31,wherein a is
 0. 35. A compound according to claim 31, wherein R₆ is Cl.36. A compound according to claim 31, wherein R₅ and R₆ are both Cl. 37.A compound according to claim 31, wherein c and e are both
 0. 38. Acompound according to claim 31, wherein c is 0 and e is
 1. 39. Acompound according to claim 31, wherein each R₇ is H.
 40. A compoundaccording to claim 35, wherein R₅ is selected from the group consistingof

wherein the subscript r is an integer of from 2 to 4, and the subscripts is an integer of from 1 to
 6. 41. A compound according to claim 35,wherein R₅ is N(R₇)₂ and the two R₇ groups are linked together to form a4, 5, 6, or 7 member ring.
 42. A compound of claim 1, represented byformula (V):

wherein R₅ is a member selected from the group consisting of halogen,OR₇ and N(R₇)₂; each R₇ is a member independently selected from thegroup consisting of H, a substituted or unsubstituted (C₁-C₁₂)alkylgroup and a substituted or unsubstituted (C₁-C₁₂)heteroalkyl group; m is2, 3, or 4; and n is 0 or 1; with the proviso that at least one of R₅and R₇ is a positively charged group.
 43. A compound of claim 1,represented by the formula (VI):

wherein R₅ is a member selected from the group consisting of halogen,OR₇ and N(R₇)₂; each R₇ is independently selected from the groupconsisting of H, a substituted or unsubstituted (C₁-C₁₂)alkyl group anda substituted or unsubstituted (C₁-C₁₂)heteroalkyl group; b is 1, 2, 3or 4; and f is 0 or 1; with the proviso that at least one of R₅ and R₇is a positively charged group.
 44. A compound of claim 1, represented bythe formula (VII):


45. A compound of claim 1, represented by the formula (VIII):


46. A compound of claim 1, represented by the formula (IX):


47. A compound of claim 1, represented by the formula (X):


48. A compound of claim 1, represented by the formula (XI):


49. A composition comprising a pharmaceutically acceptable carrier andan anti-infective amount of a compound of claim
 1. 50. A compositioncomprising a pharmaceutically acceptable carrier and an anti-infectiveamount of a compound of claim
 31. 51. A method of inhibiting pathogenproliferation, comprising contacting a pathogen of a eukaryotic organismwith a proliferation-inhibiting amount of a compound of claim
 1. 52. Amethod of inhibiting pathogen proliferation, comprising contacting apathogen of a eukaryotic organism with a proliferation-inhibiting amountof a compound of claim
 31. 53. A method according to claim 58 whereinthe pathogen is selected from the group consisting of Staphylococcusspp., Streptococcus spp., Enterococcus spp., Corynebacterium spp.,Listeria spp., Bacillus spp., Micrococcus spp., Peptostreptococcus spp,Clostridium spp., Propionibacterium spp., Escherichia spp., Pseudomonasspp., Haemophilus spp., Candida spp., Cryptococcus spp., Aspergillusspp., Trichophyto spp., Paecilomyces spp., Saccharomyces spp., Fusariumspp., Acinetobacter spp., Alcaligenes spp., Campylobacter spp.,Citrobacter spp., Enterobacter spp., Proteus spp., Salmonella spp.,Shigella spp., Helicobacter spp., Neisseria spp., Vibrio spp.,Bacteroides spp., Prevotella spp., Mycoplasma spp., Mycobacteria spp.,and Clamydia spp.